Novel kinases and uses thereof

ABSTRACT

Novel kinase polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length kinase proteins, the invention further provides isolated kinase fusion proteins, antigenic peptides, and anti-kinase antibodies. The invention also provides kinase nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a kinase gene has been introduced or disrupted. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/345,473, filed Jun. 30, 1999, herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to novel kinase nucleic acid sequences andproteins. Also provided are vectors, host cells, and recombinant methodsfor making and using the novel molecules.

BACKGROUND OF THE INVENTION

Phosphate tightly associated with a molecule, e.g., a protein, has beenknown since the late nineteenth century. Since then, a variety ofcovalent linkages of phosphate to proteins have been found. The mostcommon involve esterification of phosphate to serine, threonine, andtyrosine with smaller amounts being linked to lysine, arginine,histidine, aspartic acid, glutamic acid, and cysteine. The occurrence ofphosphorylated molecules, e.g., proteins, implies the existence of oneor more kinases, e.g., protein kinases, capable of phosphorylatingvarious molecules, e.g., amino acid residues on proteins, and also ofphosphatases, e.g., protein phosphatases, capable of hydrolyzing variousphosphorylated molecules, e.g., phosphorylated amino acid residues onproteins.

Protein kinases play critical roles in the regulation of biochemical andmorphological changes associated with cellular growth and division(D'Urso et al. (1990) Science 250:786-791; Birchmeier et al. (1993)Bioessays 15:185-189). They serve as growth factor receptors and signaltransducers and have been implicated in cellular transformation andmalignancy (Hunter et al. (1992) Cell 70:375-387; Posada et al. 1992)Mol. Biol. Cell 3:583-592; Hunter et al. (1994) Cell 79:573-582). Forexample, rotein kinases have been shown to participate in thetransmission of signals from growth-factor receptors (Sturgill et al.(1988) Nature 344:715-718; Gomez et al. (1991) Nature 353:170-173),control of entry of cells into mitosis (Nurse (1990) Nature 344:503-508;Maller (1991) Curr. Opin. Cell Biol. 3:269-275) and regulation of actinbundling (Husain-Chishti et al. (1988) Nature 334:718-721).

Protein kinases can be divided into different groups based on eitheramino acid sequence similarity or specificity for eitherserine/threonine or tyrosine residues. A small number ofdual-specificity kinases have also been described. Within the broadclassification, kinases can be further subdivided into families whosemembers share a higher degree of catalytic domain amino acid sequenceidentity and also have similar biochemical properties. Most proteinkinase family members also share structural features outside the kinasedomain that reflect their particular cellular roles. These includeregulatory domains that control kinase activity or interaction withother proteins (Hanks et al. (1988) Science 241:42-52).

Kinases play critical roles in cellular growth. Therefore, novel kinasepolynucleotides and proteins are useful for modulating cellular growth,differentiation and/or development.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to kinase nucleic acidsequences are provided. Additionally amino acid sequences correspondingto the polynucleotides are encompassed. In particular, the presentinvention provides for isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences shown in SEQ IDNOs:2, 4, 6, 8, 10, 12, and 14. Further provided are kinase polypeptideshaving an amino acid sequence encoded by a nucleic acid moleculedescribed herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The kinase molecules of the present invention are useful for modulatingcellular growth and/or cellular metabolic pathways particularly forregulating one or more proteins involved in growth and metabolism.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding kinase proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of kinase-encoding nucleic acids.

Another aspect of this invention features isolated or recombinant kinaseproteins and polypeptides. Preferred kinase proteins and polypeptidespossess at least one biological activity possessed by naturallyoccurring kinase proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention. Additionally,fragments and substantially homologous fragments of the nucleotide andamino acid sequences are provided.

Antibodies and antibody fragments that selectively bind the kinasepolypeptides and fragments are provided. Such antibodies are useful indetecting the kinase polypeptides as well as in modulating cellulargrowth and metabolism.

In another aspect, the present invention provides a method for detectingthe presence of kinase activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of kinase activity such that the presence of kinase activityis detected in the biological sample.

In yet another aspect, the invention provides a method for modulatingkinase activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) kinase activity or expression suchthat kinase activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds to kinaseprotein. In another embodiment, the agent modulates expression of kinaseprotein by modulating transcription of a kinase gene, splicing of akinase mRNA, or translation of a kinase mRNA. In yet another embodiment,the agent is a nucleic acid molecule having a nucleotide sequence thatis antisense to the coding strand of the kinase mRNA or the kinase gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant kinaseprotein activity or nucleic acid expression by administering an agentthat is a kinase modulator to the subject. In one embodiment, the kinasemodulator is a kinase protein. In another embodiment, the kinasemodulator is a kinase nucleic acid molecule. In other embodiments, thekinase modulator is a peptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding a kinase protein; (2) misregulation of a gene encoding akinase protein; and (3) aberrant post-translational modification of akinase protein, wherein a wild-type form of the gene encodes a proteinwith a kinase activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a kinase protein. Ingeneral, such methods entail measuring a biological activity of a kinaseprotein in the presence and absence of a test compound and identifyingthose compounds that alter the activity of the kinase protein.

The invention also features methods for identifying a compound thatmodulates the expression of kinase genes by measuring the expression ofthe kinase sequences in the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleotide and amino acid sequence for h12832.

FIG. 2 shows the amino acid sequence alignment for the protein (h12832;SEQ ID NO:2) encoded by human 12832 (SEQ ID NO:1) with the Arabidopsisthaliana protein kinase homologue (A. thal. protein kinase homolog;GenBank Accession Number AAB71975; SEQ ID NO:26), the Arabidopsisthaliana putative receptor kinase (A. thal. putative Rc. Kinase; GenBankAccession Number AAB71975; SEQ ID NO:27), the Arabidopsis thalianareceptor-kinase isolog (A. thal. Rc. kinase isolog; GenBank AccessionNumber AAB65490; SEQ ID NO:28), the C. elegans tyrosine kinase (C. ele.Tyr. Kinase; GenBank Accession Number AAC47047; SEQ ID NO:29) and thehuman mixed lineage kinase 1 (hMLK1; SP Accession Number P80192; SEQ IDNO:30).

FIG. 3 shows a dendrogram for the h12832 gene.

FIG. 4 provides the nucleotide and amino acid sequence for h14138.

FIG. 5 shows the amino acid sequence alignment for the protein (h14138;SEQ ID NO:4) encoded by human 14138 (SEQ ID NO:3) with the Dictyosteliumdiscoideum PkgA protein (Di. Dis. PkgA; GenBank Accession NumberAAB70848, SEQ ID NO:31), the hypothetical human serine-threonine proteinkinase R31240_(—)1 (h19p13.2; GenBank Accession Number AAB51171; SEQ IDNO:32), the human KIAA0561 protein (hKIAA0561; DBJ Accession NumberBAA20762; SEQ ID NO:33), the human KIAA0807 protein (hKIAA0807; DBJAccession Number BAA34527; SEQ ID NO:34), the mouse protein kinase(mMAST205; GenBank Accession Number AAC04132; SEQ ID NO:35) and the S.pombe protein kinase CEK1 (CEK1, SP Accession Number P38938; SEQ IDNO:36).

FIG. 6 shows a dendrogram for the h14138 gene.

FIG. 7 provides the nucleotide and amino acid sequence for h14833.

FIG. 8 shows the amino acid sequence alignment for the protein (h14833;SEQ ID NO:6) encoded by human 14833 (SEQ ID NO:5) with the avianerythroblastosis virus (strain S13) sea tyrosine-protein kinasetransforming protein (Avi. SEA Tyr. Pro. Kin.; SP Accession NumberP23049; SEQ ID NO:26), the avian erythroblastosis virus env-seapolyprotein (Avi. Tyr. Kinase env-sea; NB Accession Number A33902; SEQID NO:27), the Caenorhabditis elegans protein containing similarity toprotein kinase domains (C. ele. P. Kin. Domains; GenBank AccessionNumber AAC19211; SEQ ID NO:28), the putative fruit fly (Drosophilamelanogaster) torso tyrosine-protein kinase receptor (Dros. TOR Tyr.Kinase Rc.; SP Accession Number P18475; SEQ ID NO:29), the c-sea chicken(gallus gallus) transmembrane protein-tyrosine kinase (Gal. Proto. T.M.Tyr. Kinase; GenBank Accession Number AAA48729; SEQ ID NO:30), the Hydravulgaris receptor protein-tyrosine kinase (Hyd. Rc. Pro. Kinase; GenBankAccession Number AAA65223; SEQ ID NO:31), and the purple sea urchin(Strongylocentrotus purpuratus) fibroblast growth factor receptor (Str.FGF Rc.; GenBank Accession Number AAC47258; SEQ ID NO:32). The sequencealignment was generated using the Clustal method with PAM 250 residueweight table.

FIG. 9 shows a dendrogram for the h14833 gene.

FIG. 10 provides the nucleotide and amino acid sequence for h15590.

FIG. 11 shows the amino acid sequence alignment for the protein (h15990;SEQ ID NO:8) encoded by human 15990 (SEQ ID NO:7) with the Arabidopsisthaliana putative protein kinase (A. thal. BAC clone; GenBank AccessionNumber AAD30583; SEQ ID NO:33), the Arabidopsis thalianaserine/threonine kinase-like protein (A. thal. Ser/Thr kin-like pro; EMBAccession Number CAB43919; SEQ ID NO:34), the human serine/threoninekinase RICK (hBAC clone; GenBank Accession Number AAC24561; SEQ IDNO:35), the human serine/threonine kinase receptor interacting protein(hSer/Thr Kin. RIP; SP Accession Number Q13546; SEQ ID NO:36), themurine serine/threonine kinase receptor interacting protein (mSer/ThrPro. Kin. RIP; SP Accession Number Q60855; SEQ ID NO:39), and the Rattusnorvegicus homocysteine respondent protein (GenBank Accession NumberAAD02059; SEQ ID NO:38). The sequence alignment was generated using theClustal method.

FIG. 12 shows a dendrogram for the h15990 gene.

FIG. 13 provides the nucleotide and amino acid sequence for h15993.

FIG. 14 shows the amino acid sequence alignment for the protein (h15993;SEQ ID NO:10) encoded by human 15993 (SEQ ID NO:9) with the Arabidopsisthaliana putative MAP kinase (A. thal. MAPK; EMB Accession NumberCAB43520; SEQ ID NO:39), the Arabidopsis thaliana Ste20-like kinasehomolog (A. tha. Sete20-like Kinase homo.; GenBank Accession NumberAAC18797; SEQ ID NO:40), the Arabidopsis thaliana protein kinase-likeprotein (A. thal. cosmid; EMB Accession Number CAB41172; SEQ ID NO:41),the C. elegans protein having weak similarity with many protein kinases(C. ele. Kinase-like; EMB Accession Number CAA99887; SEQ ID NO:42), theC. elegans tyrosine-protein kinase-like protein (C. ele. Tyr.Kinase-like; EMB Accession Number CAA15621; SEQ ID NO:43), the humanputative mitogen-activated protein kinase kinase kinase (hMAPKKK; EMBAccession Number CAB44308; SEQ ID NO:44), the Oryza sativa mitogenactivated protein kinase kinase (O. sat. MEK1; GenBank Accession NumberAAC32599; SEQ ID NO:45), the Phycomyces blakesleeanus serine/threonineprotein kinase pkpa (P. bla. PKPA; SP Accession Number Q01577; SEQ IDNO:46), and the C. elegans serine/threonine protein kinase-like protein(C. ele. Ser/thr Kinase-like; EMB Accession Number CAA92591; SEQ IDNO:47). The sequence alignment was generated using the Clustal method.

FIG. 15 shows a dendrogram for the h15993 gene.

FIG. 16 provides the nucleotide and amino acid sequence for h16341.

FIG. 17 shows the amino acid sequence alignment for the protein (h16341;SEQ ID NO:12) encoded by human 16341 (SEQ ID NO:11) with the human limdomain kinase 2 (hLIK2; SP accession Number P53671; SEQ ID NO:48), thehuman lim kinase (hLim Kinase; GenBank Accession Number AAB54055; SEQ IDNO:49), the human testis-specific protein kinase 1 (hTESK; SP accessionNumber Q15569; SEQ ID NO:50), the murine lim kinase 2b (mLIMK2b; DBJAccession Number BAA24489; SEQ ID NO:51), the murine testis-specificlim-kinase 2 (mLimk2t; DBJ Accession Number BAA31147; SEQ ID NO:52), themurine testis-specific protein kinase 1 (mTESK1; DBJ Accession NumberBAA25124; SEQ ID NO:53), and mTESK1.1; DBJ Accession Number BAA25125;SEQ ID NO:54), the R. norvegicus testis-specific protein kinase 1(rTESK; SP Accession Number Q63572; SEQ ID NO:55), and Xenopus laevisLIM motif-containing protein kinase, Xlimk1 (S. lae. Xlimk1; DBJAccession Number BAA21488; SEQ ID NO:56). The sequence alignment wasgenerated using the Clustal method.

FIG. 18 shows a dendrogram for the h16341 gene.

FIG. 19 provides the nucleotide and amino acid sequence for h2252.

FIG. 20 shows the amino acid sequence alignment for the protein (h2252;SEQ ID NO:14) encoded by human 2252 (SEQ ID NO:13) with the C. elegansserine/threonine kinase (C. ele. cosmid, GenBank Accession NumberAAC69038; SEQ ID NO:57), the Dictyostelium discoideum severin kinase(Disto. Disc. Severin Kinase., GenBank Accession Number AAC24522; SEQ IDNO: 58), the human Ste20-like kinase (hSTE20-like Kinase; EMB AccessionNumber CAA67700; SEQ ID NO:59), the human Ste20-like kinase 3(hSTE20-like Kinase-3; GenBank Accession Number AAB82560; SEQ ID NO:60),the human YSK1 protein (hYSK1, DBJ Accession Number BAA20420; SEQ IDNO:61) and the mouse Ste20-like kinase (mSTE20-like Kinase; GenBankAccession Number AAD01208; SEQ ID NO:62).

FIG. 21 shows a dendrogram for the h2252 gene.

FIG. 22 shows expression of h12832 in various tissues and cell typesrelative to expression in human CD14.

FIG. 23 shows expression of h1438 in various tissues and cell typesrelative to expression in human mPB leukocytes.

FIG. 24 shows expression of h14833 in various tissues and cell typesrelative to expression in human CD14.

FIG. 25 shows expression of h15990 in various tissues and cell typesrelative to expression in human HEK 293.

FIG. 26 shows expression of h16341 in various tissues and cell typesrelative to expression in human liver fibrosis 194.

FIG. 27 shows expression of h2252 in various tissues and cell typesrelative to expression in human brain tissue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “kinase” nucleic acid andpolypeptide molecules, which play a role in, or function in, signalingpathways associated with cellular growth and/or cellular metabolicpathways. These growth and metabolic pathways are described in Lodish etal. (1995) Molecular Cell Biology (Scientific American Books Inc., NewYork, N.Y.) and Stryer Biochemistry, (W.H. Freeman, New York), thecontents of which are incorporated herein by reference. In oneembodiment, the kinase molecules modulate the activity of one or moreproteins involved in cellular growth or differentiation, e.g., cardiac,epithelial, or neuronal cell growth or differentiation. In anotherembodiment, the kinase molecules of the present invention are capable ofmodulating the phosphorylation state of a kinase molecule or thephosphorylation state of one or more proteins involved in cellulargrowth or differentiation, e.g., cardiac, epithelial, or neuronal cellgrowth or differentiation, as described in, for example, Lodish et al.and Stryer, supra. In addition, kinases of the present invention aretargets of drugs described in Goodman and Gilman (1996), ThePharmacological Basis of Therapeutics (9^(th) ed.) Hartman & LimbardEditors, the contents of which are incorporated herein by reference.Particularly, the kinases of the invention may modulate phosphorylationin tissues and cells including lymph node, spleen, thymus, brain, lung,skeletal muscle, fetal liver, tonsil, colon, heart, liver, immune cells,including T cells, Th1 and Th2 cells, leukocytes, blood marrow, etc.

As used herein, the term “kinase” includes a protein, polypeptide, orother non-proteinaceous molecule that is capable of modulating its ownphosphorylation state or the phosphorylation state of a differentprotein, polypeptide, or other non-proteinaceous molecule. Kinases canhave a specificity for (i.e., a specificity to phosphorylate)serine/threonine residues, tyrosine residues, or both serine/threonineand tyrosine residues, e.g., the dual-specificity kinases. As referredto herein, kinases such as protein kinases preferably include acatalytic domain of about 200-400 amino acid residues in length,preferably about 200-300 amino acid residues in length, or morepreferably about 250-300 amino acid residues in length, which includespreferably 5-20, more preferably 5-15, or most preferably 11 highlyconserved motifs or subdomains separated by sequences of amino acidswith reduced or minimal conservation. Specificity of a kinase forphosphorylation of either tyrosine or serine/threonine can be predictedby the sequence of two of the subdomains (VIb and VIII) in whichdifferent residues are conserved in each class (as described in, forexample, Hanks et al. (1988) Science 241:42-52, the contents of whichare incorporated herein by reference). These subdomains are alsodescribed in further detail herein.

Kinases play a role in signaling pathways associated with cellulargrowth. For example, protein kinases are involved in the regulation ofsignal transmission from cellular receptors, e.g., growth-factorreceptors, entry of cells into mitosis, and the regulation ofcytoskeleton function, e.g., actin bundling.

Assays for measuring Kinase activity are well known in the art dependingon the particular kinase. Specific assay protocols are available instandard sources known to the ordinarily skilled artisan. For example,see “Kinases” in Ausueel et al., eds. (1994-1998) Current Protocols inMolecular Biology (3) and references cited therein;http://www.sdsc.edu/Kinases/pkr/pk protocols.html; andhttp://www.sdsc.edulKinases/pkr/pk protocols/tyr synpep assay.html

Inhibition or over stimulation of the activity of kinases involved insignaling pathways associated with cellular growth can lead to perturbedcellular growth, which can in turn lead to cellular growthrelated-disorders. As used herein, a “cellular growth-related disorder”includes a disorder, disease, or condition characterized by aderegulation, e.g., an upregulation or a downregulation, of cellulargrowth. Cellular growth deregulation may be due to a deregulation ofcellular proliferation, cell cycle progression, cellular differentiationand/or cellular hypertrophy. Examples of cellular growth relateddisorders include cardiovascular disorders such as heart failure,hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathiccardiomyopathy, or angina; proliferative disorders or differentiativedisorders such as cancer, e.g., melanoma, prostate cancer, cervicalcancer, breast cancer, colon cancer, or sarcoma. Disorders associatedwith the following cells or tissues are also encompassed: lymph node,spleen, thymus, brain, lung, skeletal muscle, fetal liver, tonsil,colon, heart, liver, immune cells, including T cells, Th1 and Th2 cells,leukocytes, blood marrow, etc. The compositions are also useful for thetreatment of liver fibrosis and other liver-related disorders.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of immune, inflammatory,respiratory, and hematological disorders. Such immune disorders include,but are not limited to, chronic inflammatory diseases and disorders,such as Crohn's disease, reactive arthritis, including Lyme disease,insulin-dependent diabetes, organ-specific autoimmunity, includingmultiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis), certain viralinfections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

Respiratory disorders include, but are not limited to, apnea, asthma,particularly bronchial asthma, berillium disease, bronchiectasis,bronchitis, bronchopneumonia, cystic fibrosis, diphtheria, dyspnea,emphysema, chronic obstructive pulmonary disease, allergicbronchopulmonary aspergillosis, pneumonia, acute pulmonary edema,pertussis, pharyngitis, atelectasis, Wegener's granulomatosis,Legionnaires disease, pleurisy, rheumatic fever, and sinusitis.

Hematologic disorders include but are not limited to anemias includingsickle cell and hemolytic anemia, hemophilias including types A and B,leukemias, thalassemias, spherocytosis, Von Willebrand disease, chronicgranulomatous disease, glucose-6-phosphate dehydrogenase deficiency,thrombosis, clotting factor abnormalities and deficiencies includingfactor VIII and IX deficiencies, hemarthrosis, , hematemesis, hematomas,hematuria, hemochromatosis, hemoglobinuria, hemolytic-uremic syndrome,thrombocytopenias including HIV-associated thrombocytopenia, hemorrhagictelangiectasia, idiopathic thrombocytopenic purpura, thromboticmicroangiopathy, hemosiderosis.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as kinase protein and nucleic acidmolecules, that comprise a family of molecules having certain conservedstructural and functional features. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain or motif and having sufficient amino acid ornucleotide sequence identity as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin, as well as other, distinct proteins of human origin oralternatively, can contain homologues of non-human origin. Members of afamily may also have common functional characteristics.

One embodiment of the invention features kinase nucleic acid molecules,preferably human kinase molecules, that were identified based on aconsensus motif or protein domain characteristic of a kinase family ofproteins. Specifically, seven novel human genes, termed clones h12832,h14138 (partial-length), h14833, h15990 (partial length), 15993 (partiallength), h16341 (partial length), and h2252, are provided. Suchsequences are referred to as “kinase” sequences indicating that thegenes and the partial gene sequences share sequence similarity withkinase genes. The kinases of the invention fall within the eukaryoticprotein kinase family.

A. The Eukaryotic Protein Kinase Nucleic Acid and Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode eukaryotic protein kinase polypeptides. Eukaryoticprotein kinases (described in, for example, Hanks et al. (1995) FASEB J.9:576-596) are enzymes that belong to an extensive family of proteinsthat share a conserved catalytic core common to both serine/threonineand tyrosine protein kinases. There are a number of conserved regions inthe catalytic domain of protein kinases. One of these regions, locatedin the N-terminal extremity of the catalytic domain, is a glycine-richstretch of residues in the vicinity of a lysine residue, which has beenshown to be involved in ATP binding. Another region, located in thecentral part of the catalytic domain, contains a conserved aspartic acidresidue which is important for the catalytic activity of the enzyme(Knighton et al. (1991) Science 253:407-414). Two signature patternshave been described for this region: one specific for serine/threoninekinases and one for tyrosine kinases.

Eukaryotic protein kinase polypeptides of the present inventionpreferably include one of the following consensus sequences:[LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIVCAT]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIV P]-[LIVMFAGCKR]-K [Kbinds ATP] [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT] (3) [D isan active site residue][LIVMFYC]-x-[HY]-x-D-[LIVMFY]-[RSTAC]-x(2)-N-[LIVM FYC](3) [D is anactive site residue]B. The Adenylate Kinase Nucleic Acid and Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode adenylate kinase polypeptides. Adenylate kinase (AK)(described in Schulz (1987) Cold Spring Harbor Symp. Quant. Biol.52:429-439) is a monomeric enzyme that catalyzes the reversible transferof MgATP to AMP (MgATP+AMP=MgADP+ADP).

In mammals there are three different isozymes: AK1 (or myokinase), whichis cytosolic; AK2, which is located in the outer compartment ofmitochondria; and AK3 (or GTP:AMP phosphotransferase), which is locatedin the mitochondrial matrix and which uses MgGTP instead of MgATP.

Several regions of AK family enzymes are well conserved, including theATP-binding domains. This region includes an aspartic acid residue thatis part of the catalytic cleft of the enzyme and is involved in a saltbridge.

It also includes an arginine residue whose modification leads toinactivation of the enzyme. Adenylate kinase polypeptides of the presentinvention preferably include the following consensus sequence:[LIVMFYW](3)-D-G-[FYI]-P-R-x(3)-[NQ]C. The Guanylate Kinase Nucleic Acid and Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode guanylate kinase polypeptides. Guanylate kinase(described in Stehle (1992) J. Mol. Biol. 224:1127-1141) catalyzes theATP-dependent phosphorylation of GMP into GDP and it is essential forrecycling GMP and indirectly, cGMP. In prokaryotes (such as Escherichiacoli), lower eukaryotes (such as yeast) and in vertebrates, guanylatekinase is a highly conserved monomeric protein of about 200 amino acids.

Guanylate kinases are characterized by the presence of one or more of aDHR domain, and an SH3 domain (Woods et al. (1994) Mech. Dev. 44:85-89).There is also an ATP-binding site (P-loop) in the N-terminal section ofguanylate kinases. Guanylate kinase polypeptides of the presentinvention contain a highly conserved region that contains two arginineresidues and a tyrosine residue, which are involved in GMP-binding. Thisconserved region is shown below:T-[ST]-R-x(2)-[KR]-x(2)-[DE]-x(2)-G-x(2)-Y-x-[FY]- [LIVMK]D. The Pyruvate Kinase Nucleic Acid and Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode pyruvate kinase polypeptides. Pyruvate kinase (PK)(described in Muirhead (1990) Biochem. Soc. Trans. 18:193-196) catalyzesthe final step in glycolysis, the conversion of phosphoenolpyruvate topyruvate with the concomitant phosphorylation of ADP to ATP. PK requiresboth magnesium and potassium ions for its activity. PK is found in allliving organisms. In vertebrates there are four, tissue-specificisozymes: (L) liver, R (red cells), M1 (muscle, heart, and brain), andM2 (early fetal tissues).

All PK isozymes appear to be tetramers of identical subunits of about500 amino acid residues. PKs contain a conserved region that includes alysine residue that appears to be the acid/base catalyst responsible forthe interconversion of pyruvate and enolpyruvate, and a glutamic acidresidue implicated in the binding of the magnesium ion.

The pyruvate kinase polypeptides of the present invention preferablyinclude the following consensus sequence:[LIVAC]-x-[LIVM](2)-[SAPCV]-K-[LIV]-E-[NKRST]-x-[D EQH]-[GSTA]-[LIVM] [Kis the active site residue] [E is a magnesium ligand]

-   -   [K is the active site residue] [E is a magnesium ligand]        E. The Phosphatidylinositol-3 Kinase Nucleic Acid and        Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode phosphatidylinositol 3-kinase polypeptides.Phosphatidylinositol 3-kinase (PI3-kinase) (described in Hiles et al.(1992) Cell 70:419-429) is an enzyme that phosphorylatesphosphoinositides on the 3-hydroxyl group of the inositol ring.

The three products of PI3-kinase [PI-3-P, PI-3,4-P(2), andPI-3,4,5-P(3)] function as second messengers in cell signaling. Themammalian P13 kinase is a heterodimer of a 110 kDa catalytic chain(p110) and an 85 kDa subunit (p85) which allows it to bind to activatedtyrosine protein kinases. The PI3-kinases share a well conserved domainat their C-terminal section (Kunz et al. (1993) Cell 73:585-596).

The phosphatidylinositol 3-kinase polypeptides of the present inventionpreferably include the following consensus domains:[LIVMFAC]-K-x(1,3)-[DEA]-[DE]-[LIVMC]-R-Q-[DE]-x(4)-Q[GS]-x-[AV]-x(3)-[LIVM]-x(2)-[FYH]-[LIVM](2)-x-[LIVMF]-x-D-R-H-x(2)-NNovel Kinase Sequences

The kinase genes and partial gene sequences of the invention wereidentified in a variety of cell or tissue libraries including Th2 celllibrary (h12832, h15993, h16341); natural killer T cell library (h14833,h2252); microvascular endothelial cells (h14138); mixed lymphocytereaction (h15990). The first of these clones, h12832, encodes anapproximately 1.6 kb mRNA transcript having the corresponding cDNAsequence set forth in SEQ ID NO:1. This transcript has a 966 nucleotideopen reading frame (nucleotides 191-1156 of SEQ ID NO:1), which encodesa 322 amino acid protein (SEQ ID NO:2). The molecule may havetransmembrane segments from amino acids (aa) 19-35 and 230-250 aspredicted by MEMSAT. Prosite program analysis was used to predictvarious sites within the h12832 protein. N-glycosylation sites werepredicted at aa 196-199 and 249-252. A cAMP- and cGMP-dependent proteinkinase phosphorylation site was predicted at aa 16-19. Protein kinase Cphosphorylation sites were predicted at aa 14-16, 52-54, 181-183, and225-227. Casein kinase II phosphorylation sites were predicted at aa122-125, 198-201, 236-239, 251-254, 260-263, 264-267, and 301-304.N-myristoylation sites were predicted at aa 41-46 and 118-123. Aserine/threonine protein kinase active-site signature sequence waspredicted at aa 163-175. The h12832 protein possesses a eukaryoticprotein kinase domain, from aa 32 to aa 316, as predicted by HMMer,Version 2. Within this domain, critical residues are conserved at aminoacid (aa) positions 39, 41, 46, 62, 64, 85, 94, 96, 116, 123, 153,156-158, 165, 167, 169, 172, 183, 186, 188, 208, 209, 216, 229, 234,237, 239, 246, 296, 304, 305, and 308. Critical residues are missing ataa positions 166, 187, 214, and 215, while important residues aremissing at aa positions 44, 121, 149, 173, 174, 212, 231, 232, and 284.“Critical residues” are those residues that are recognized as importantfor activity and are highly conserved in known kinases. “Importantresidues” are those residues generally conserved among kinases.

The h12832 protein shares similarity with several protein kinases (seeFIG. 2). Dendrogram analysis of this gene indicates it shares closesthomology with C. elegans tyrosine kinase (C. ele. Tyr. Kinase; GenBankAccession Number AAC47047; SEQ ID NO:29). The h12832 protein sharesapproximately 26% identity with this protein kinase receptor asdetermined by pairwise alignment (see Needleman and Wunsch (1970) J.Mol. Biol. 48:444). (see FIG. 3).

The partial gene sequence designated clone h14138 encodes anapproximately 0.83 kb transcript mRNA having the corresponding cDNA setforth in SEQ ID NO:3. This transcript has a 522 nucleotide open readingframe (nucleotides 1-522 of SEQ ID NO:3), which encodes a 174 amino acidpolypeptide (SEQ ID NO:4). An analysis of the disclosed h14138polypeptide sequence (SEQ ID NO:4) using the MEMSAT program predicts atransmembrane segment from aa 56-73. Prosite program analysis was alsoused to predict various sites within this partial-length proteinsequence. Protein kinase C phosphorylation sites were predicted at aa12-14, 17-19, 20-22, and 116-118. Casein kinase II phosphorylation siteswere predicted at aa 12-15, 105-108, 112-115, 131-134, and 156-159. AnN-myristoylation site was predicted at aa 9-14. The partial-lengthh14138 protein possesses a eukaryotic protein kinase domain, from aa35-130, and a protein kinase C terminal domain, from aa 131-159, aspredicted by HMMer Version 2.

The partial length h14138 protein displays similarity to several proteinkinases (see FIG. 5). Dendrogram analysis of this gene indicates itshares closest homology with human KIAA0807 protein; DBJ AccessionNumber BAA34527; SEQ ID NO: 34). The h14138 protein shares approximately35% identity with this protein kinase receptor as determined by pairwisealignment (see Needleman and Wunsch (1970) J. Mol. Biol. 48:444). (seeFIG. 6).

The third novel gene, designated clone h14833, encodes an approximately2.1 kb transcript mRNA having the corresponding cDNA set forth in SEQ IDNO:5. This transcript has a 627 nucleotide open reading fiame(nucleotides 154-780 of SEQ ID NO:5), which encodes a 209 amino acidprotein (SEQ ID NO:6) having a molecular weight of approximately 23.8kDa. An analysis of the full-length h14833 protein sequence using theMEMSAT program predicted no transmembrane domains. Prosite programanalysis of this protein predicted an N-glycosylation site at amino acid(aa) 205-208 and a cAMP- and cGMP-dependent protein kinasephosphorylation site at aa 133-136. Protein kinase C phosphorylationsites were predicted at aa 11-13, 51-53, 91-93, and 159-161. Caseinkinase II phosphorylation sites were predicted at aa 121-124, 159-162,and 173-176. N-myristoylation sites were predicted at aa 56-61 and67-72. A yrosine protein kinase specific active-site signature waspredicted at aa 34-46. The h14833 protein possesses a eukaryotic proteinkinase domain from aa 10 to aa 163.

The h14833 protein displays similarity to several protein kinases (seeFIG. 8 f. endrogram analysis of this gene indicates that the encodedh14833 protein shares closest homology with a putative fruit fly(Drosophila melanogaster) torso tyrosine-protein kinase receptor (SPAccession Number P18475; SEQ ID NO:18) (see FIG. 9). The h14833 proteinshares approximately 34% identity over a 209 amino-acid overlap withthis protein kinase receptor as determined by pairwise alignment (seeNeedleman and Wunsch (1970) J. Mol. Biol. 48:444).

The partial gene sequence designated clone h15990 encodes anapproximately 1.7 kb transcript mRNA having the corresponding cDNA setforth in SEQ ID NO:7. This transcript has a 1491 nucleotide open readingframe (nucleotides 2-1492 of SEQ ID NO:7), which encodes a 497 aminoacid polypeptide (SEQ ID NO:8). An analysis of the partial-length h15990protein sequence using the MEMSAT program predicted no transmembranedomains. Prosite program analysis of this partial-length proteinpredicted N-glycosylation sites at amino acids (aa) 78-81, 412-415,433-436, and 493-496. Glycosaminoglycan attachment sites were predictedat aa 151-154, 299-302, and 461-464. cAMP- and cGMP-dependent proteinkinase phosphorylation sites were predicted at aa 175-178 and 292-295.Protein kinase C phosphorylation sites were predicted at aa 279-281,290-292, 348-350, 382-384, 414-416, 424-426,461-463, and 495-497. Caseinkinase II phosphorylation sites were predicted at aa 179-182, 220-223,254-257, 279-282, 295-298, 304-307, 318-321, and 366-369.N-myristoylation sites were predicted at aa 9-14, 79-84, 147-152,159-164, 300-305, 402-407, 410-415, 436-441, and 457-462. A proteinkinase ATP-binding region signature sequence was predicted at aa 6-14. Aserine/threonine protein kinase active-site signature sequence waspredicted at aa 117-129. The partial-length h15990 protein possesses aeukaryotic protein kinase domain, from aa 1-259, as predicted by HMMerVersion 2.

The partial-length h15990 protein displays similarity to several proteinkinases (see FIG. 11). Dendrogram analysis of this gene indicates thatthe encoded h15990 protein shares closest homology with a Rattusnorvegicus homocysteine respondent protein (GenBank Accession NumberAAD02059; SEQ ID NO:27) (see FIG. 12). The partial-length h15990 proteinshares approximately 74.3% identity over a 142 amino-acid overlap withthis homocysteine respondent protein as determined by pairwisealignment.

The partial gene sequence designated clone h15993 encodes anapproximately 0.98 kb transcript mRNA having the corresponding cDNA setforth in SEQ ID NO:9. This transcript has a 978 nucleotide open readingframe (nucleotides 2-979 of SEQ ID NO:9), which encodes a 326 amino acidpolypeptide (SEQ ID NO:10) having a molecular weight of approximately37.2 kDa. Transmembrane segments from amino acids (aa) 168-185 and247-263 were predicted by MEMSAT. Prosite program analysis of thepartial-length h15993 protein predicted an N-glycosylation site at aa186-189, and a cAMP and cGMP-dependent protein kinase phosphorylationsite at aa 304-307. Protein kinase C phosphorylation sites werepredicted at aa 141-143 and 149-151. Casein kinase II phosphorylationsites were predicted at aa 33-36, 100-103, 246-249, 267-270, and284-287. N-myristoylation sites were predicted at aa 58-63 and 185-190.The partial-length h15993 protein possesses two eukaryotic proteinkinase domains, from aa 108 to 203 and from aa 283 to 314, as predictedby HMMer Version 2.

The partial-length h15993 protein displays similarity to several proteinkinases (see FIG. 14). Dendrogram analysis of this gene indicates thatthe encoded h15993 protein shares closest homology with a C. eleganstyrosine-protein kinase-like protein (EMB Accession Number CAA15621; SEQID NO:32) (see FIG. 15). The partial-length h15993 protein sharesapproximately 45.1% identity over a 231 amino-acid overlap with thistyrosine-protein kinase-like protein as determined by pairwisealignment.

The partial gene sequence designated clone h16341 encodes anapproximately 0.52 kb transcript mRNA having the corresponding cDNA setforth in SEQ ID NO:9. This transcript has a 516 nucleotide open readingframe (nucleotides 2-517 of SEQ ID NO:9), which encodes a 172 amino acidpolypeptide (SEQ ID NO:10) having a molecular weight of approximately19.5 kDa. An analysis of the partial-length h16341 protein sequenceusing the MEMSAT program predicted no transmembrane domains. Prositeprogram analysis of this partial-length protein predicted anN-glycosylation site at amino acids (aa) 27-30, and protein kinase Cphosphorylation sites at aa 38-40, 89-91, and 147-149. Casein kinase IIphosphorylation sites were predicted at aa 13-16 and 50-53.N-myristoylation sites were predicted at aa 20-25, 77-82, and 120-125. Aprotein kinase ATP-binding region signature sequence was predicted at aa60-68. The partial-length h16341 protein possesses a eukaryotic proteinkinase domain, from aa 58 to aa 172, as predicted by HMMer Version 2.

The partial-length h16341 protein displays similarity to several proteinkinases (see FIG. 17). Dendrogram analysis of this gene indicates thatthe encoded partial-length h16341 protein shares closest homology with amurine testis-specific protein kinase 1 (DBJ Accession Number BAA25124;SEQ ID NO:42) (see FIG. 18). The partial-length h16341 protein sharesapproximately 91.7% identity over a 172 amino-acid overlap with thistyrosine-protein kinase-like protein as determined by pairwisealignment.

The last of these novel genes, designated clone h2252, encodes anapproximately 1.7 kb transcript mRNA having the corresponding cDNA setforth in SEQ ID NO:13. This transcript has a 1248 nucleotide openreading frame (nucleotides 275-1522 of SEQ ID NO:13), which encodes a416 amino acid protein (SEQ ID NO:14). Transmembrane segments werepredicted at aa 95-111 and 346-362 using the MEMSAT program. Prositeanalysis of the full-length h2252 protein predicted N-glycosylationsites at aa 44-47, 318-321, and 371-374. cAMP- and cGMP-dependentprotein kinase phosphorylation sites were predicted at aa 175-178 and aa279-282. Protein kinase C phosphorylation sites were predicted at aa137-139, 246-248, 260-262, 264-266, 278-280, 314-316, 328-330, and396-398. Casein kinase II phosphorylation sites were predicted at aa25-28, 34-37, 75-78, 106-109, 194-197, 198-201, 208-211, 246-249,264-267, 300-303, 304-307, 309-312, 314-317, 320-323, and 411-414.N-myristoylation sites were predicted at aa 12-17 and 103-108. A proteinkinase ATP-binding region signature sequence was predicted at aa 30-38.The h2252 protein possesses a eukaryotic protein kinase domain, from aa24-274, and a phosphofructokinase domain, from aa 385-406, as predictedby HMMer Version 2.0.

The partial length h2252 protein displays similarity to several proteinkinases (see FIG. 20). Dendrogram analysis of this gene indicates itshares closest homology with the human Ste20-like kinase 3 (hSTE20-likeKinase-3; GenBank Accession Number AAB82560; SEQ ID NO:60). The h2252protein shares approximately 73% identity with this protein kinasereceptor as determined by pairwise alignment (see Needleman and Wunsch(1970) J. Mol. Biol. 48:444). (see FIG. 21).

Preferred kinase polypeptides of the present invention have an aminoacid sequence sufficiently identical to the amino acid sequence of SEQID NO:2, 4, 6, 8, 10, 12, or 14, or a domain thereof. The term“sufficiently identical” is used herein to refer to a first amino acidor nucleotide sequence that contains a sufficient or minimum number ofidentical or equivalent (e.g., with a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences have acommon structural domain and/or common functional activity. For example,amino acid or nucleotide sequences that contain a common structuraldomain having at least about 45%, 55%, or 65% identity, preferably 75%identity, more preferably 85%, 95%, or 98% identity are defined hereinas sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, nonlimitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to kinase nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to kinaseprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively,PSI-Blast can be used to perform an iterated search that detects distantrelationships between molecules. When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another preferred, example of an algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0),which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. A preferred program is the Pairwise Alignment Program(Sequence Explorer), using default parameters.

Accordingly, another embodiment of the invention features isolatedkinase proteins and polypeptides having a kinase protein activity. Asused interchangeably herein, a “kinase protein activity”, “biologicalactivity of a kinase protein”, or “functional activity of a kinaseprotein” refers to an activity exerted by a kinase protein, polypeptide,or nucleic acid molecule on a kinase-responsive cell as determined invivo, or in vitro, according to standard assay techniques. A kinaseactivity can be a direct activity, such as an association with or anenzymatic activity on a second protein, or an indirect activity, such asa cellular signaling activity mediated by interaction of the kinaseprotein with a second protein. In a preferred embodiment, a kinaseactivity includes at least one or more of the following activities: (1)modulating (stimulating and/or enhancing or inhibiting) cellularproliferation, growth and/or metabolism (e.g. in those cells in whichthe sequence is expressed, including, lymph node, spleen, thymus, brain,lung, skeletal muscle, fetal liver, tonsil, colon, heart, liver, immunecells, including Th1, Th2, T cells, natural killer T cells, lymphocytes,leukocytes, blood marrow, etc.); (2) the regulation of transmission ofsignals from cellular receptors, e.g., growth factor receptors; (3) themodulation of the entry of cells into mitosis; (4) the modulation ofcellular differentiation; (5) the modulation of cell death; and (6) theregulation of cytoskeleton function, e.g., actin bundling.

An “isolated” or “purified” kinase nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein-encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated”

when used to refer to nucleic acid molecules excludes isolatedchromosomes. For example, in various embodiments, the isolated kinasenucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. A kinase protein that is substantially free ofcellular material includes preparations of kinase protein having lessthan about 30%, 20%, 10%, or 5% (by dry weight) of non-kinase protein(also referred to herein as a “contaminating protein”). When the kinaseprotein or biologically active portion thereof is recombinantlyproduced, preferably, culture medium represents less than about 30%,20%, 10%, or 5% of the volume of the protein preparation. When kinaseprotein is produced by chemical synthesis, preferably the proteinpreparations have less than about 30%, 20%, 10%, or 5% (by dry weight)of chemical precursors or non-kinase chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding kinase proteins or biologicallyactive portions thereof, as well as nucleic acid molecules sufficientfor use as hybridization probes to identify kinase-encoding nucleicacids (e.g., kinase mRNA) and fragments for use as PCR primers for theamplification or mutation of kinase nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

Nucleotide sequences encoding the kinase proteins of the presentinvention include sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11,and 13, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequences for the kinase proteins encoded by these nucleotide sequencesare set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14, respectively.

Nucleic acid molecules that are fragments of these kinase nucleotidesequences are also encompassed by the present invention. By “fragment”is intended a portion of the nucleotide sequence encoding a kinaseprotein of the invention. A fragment of a kinase nucleotide sequence mayencode a biologically active portion of a kinase protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a kinaseprotein can be prepared by isolating a portion of one of the kinasenucleotide sequences of the invention, expressing the encoded portion ofthe kinase protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the kinase protein.Generally, nucleic acid molecules that are fragments of a kinasenucleotide sequence comprise at least 15, 20, 50, 75, 100, 325, 350,375, 400, 425, 450 or 500 nucleotides, or up to the number ofnucleotides present in a full-length kinase nucleotide sequencedisclosed herein (for example, 1586, 831, 2060, 1697, 981, 518 or 1737nucleotides for SEQ ID NO:1, 3, 5, 7, 9, 11, or 13, respectively)depending upon the intended use.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if afragment is disclosed prior to the present invention, that fragment isnot intended to be encompassed by the invention. When a sequence is notdisclosed prior to the present invention, an isolated nucleic acidfragment is at least about 12, 15, 20, 25, or 30 contiguous nucleotides.Other regions of the nucleotide sequence may comprise fragments ofvarious sizes, depending upon potential homology with previouslydisclosed sequences.

A fragment of a kinase nucleotide sequence that encodes a biologicallyactive portion of a kinase protein of the invention will encode at least15, 25, 30, 50, 75, 100, 125, 150, 160, or 170 contiguous amino acids,or up to the total number of amino acids present in a full-length kinaseprotein of the invention (for example, 322, 174, 209, 503, 326, 172, or416 amino acids for SEQ ID NO:2, 4, 6, 8, 10, 12, or 14, respectively).Fragments of a kinase nucleotide sequence that are useful ashybridization probes for PCR primers generally need not encode abiologically active portion of a kinase protein.

Nucleic acid molecules that are variants of the kinase nucleotidesequences disclosed herein are also encompassed by the presentinvention. “Variants” of the kinase nucleotide sequences include thosesequences that encode the kinase proteins disclosed herein but thatdiffer conservatively because of the degeneracy of the genetic code.These naturally-occurring allelic variants can be identified with theuse of well-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically-derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the kinase proteins disclosed in thepresent invention as discussed below. Generally, nucleotide sequencevariants of the invention will have at least 45%, 55%, 65%, 75%, 85%,95%, or 98% identity to the nucleotide sequences disclosed herein. Avariant kinase nucleotide sequence will encode a kinase protein that hasan amino acid sequence having at least 45%, 55%, 65%, 75%, 85%, 95%, or98% identity to an amino acid sequence of a kinase protein disclosedherein.

In addition to the kinase nucleotide sequences shown in SEQ ID NOs:1, 3,5, 7, 9, 11, and 13, it will be appreciated by those skilled in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of kinase proteins may exist within a population (e.g., thehuman population). Such genetic polymorphism in a kinase gene may existamong individuals within a population due to natural allelic variation.An allele is one of a group of genes that occur alternatively at a givengenetic locus. As used herein, the terms “gene” and “recombinant gene”refer to nucleic acid molecules comprising an open reading frameencoding a kinase protein, preferably a mammalian kinase protein. Asused herein, the phrase “allelic variant” refers to a nucleotidesequence that occurs at a kinase locus or to a polypeptide encoded bythe nucleotide sequence. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of the kinase gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations in a kinase sequence that are the result ofnatural allelic variation and that do not alter the functional activityof kinase proteins are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding kinase proteins from otherspecies (kinase homologues), which have a nucleotide sequence differingfrom that of the kinase sequences disclosed herein, are intended to bewithin the scope of the invention. Nucleic acid molecules correspondingto natural allelic variants and homologues of the kinase cDNAs of theinvention can be isolated based on their identity to the mouse kinasenucleic acids disclosed herein using the mouse cDNAs, or a portionthereof, as a hybridization probe according to standard hybridizationtechniques under stringent hybridization conditions as disclosed below.

In addition to naturally-occurring allelic variants of the kinasesequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of the invention thereby leading to changes in theamino acid sequence of the encoded kinase protein, without altering thebiological activity of the kinase protein. Thus, an isolated nucleicacid molecule encoding a kinase protein having a sequence that differsfrom that of SEQ ID NO:2, 4, 6, 8, 10, 12, or 14 can be created byintroducing one or more nucleotide substitutions, additions, ordeletions into the nucleotide sequences disclosed herein, such that oneor more amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of a kinase protein (e.g., thesequence of SEQ ID NO:2, 4, 6, 8, 10, 12, or 14) without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Such substitutions would not bemade for conserved amino acid residues or for amino acid residuesresiding within a conserved protein domain, such as the criticaleukaryotic protein kinase domain of all disclosed clones, and thephosphofructo-kinase domain of clone h2252, where such residues areessential for protein activity.

Alternatively, variant kinase nucleotide sequences can be made byintroducing mutations randomly along all or part of a kinase codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for kinase biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Thus the nucleotide sequences of the invention include those sequencesdisclosed herein as well as fragments and variants thereof. The kinasenucleotide sequences of the invention, and fragments and variantsthereof, can be used as probes and/or primers to identify and/or clonekinase homologues in other cell types, e.g., from other tissues, as wellas kinase homologues from other mammals. Such probes can be used todetect transcripts or genomic sequences encoding the same or identicalproteins. These probes can be used as part of a diagnostic test kit foridentifying cells or tissues that misexpress a kinase protein, such asby measuring levels of a kinase-encoding nucleic acid in a sample ofcells from a subject, e.g., detecting kinase mRNA levels or determiningwhether a genomic kinase gene has been mutated or deleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).Kinase nucleotide sequences isolated based on their sequence identity tothe kinase nucleotide sequences set forth herein or to fragments andvariants thereof are encompassed by the present invention.

In a hybridization method, all or part of a known kinase nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). The so-called hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragrnents, or other oligonucleotides,and may be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known kinasenucleotide sequences disclosed herein. Degenerate primers designed onthe basis of conserved nucleotides or amino acid residues in a knownkinase nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of akinase nucleotide sequence of the invention or a fragment or variantthereof. Preparation of probes for hybridization is generally known inthe art and is disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

For example, in one embodiment, a previously unidentified kinase nucleicacid molecule hybridizes under stringent conditions to a probe that is anucleic acid molecule comprising one of the kinase nucleotide sequencesof the invention or a fragment thereof. In another embodiment, thepreviously unknown kinase nucleic acid molecule is at least 300, 325,350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 2,000,3,000, or 4,000 nucleotides in length and hybridizes under stringentconditions to a probe that is a nucleic acid molecule comprising one ofthe kinase nucleotide sequences disclosed herein or a fragment thereof.

Accordingly, in another embodiment, an isolated previously unknownkinase nucleic acid molecule of the invention is at least 300, 325, 350,375, 400, 425, 450, 500, 518, 550, 600, 650, 700, 800, 831, 900, 981,1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900,2,000, or 2,060 nucleotides in length and hybridizes under stringentconditions to a probe that is a nucleic acid molecule comprising one ofthe nucleotide sequences of the invention, preferably the codingsequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, or 13, or acomplement, fragment, or variant thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences having at least 60%, 65%, 70%, preferably 75%identity to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology (John Wiley & Sons, NewYork (1989)), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. In another preferredembodiment, stringent conditions comprise hybridization in 6×SSC at 42°C., followed by washing with 1×SSC at 55° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to akinase sequence of the invention corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein).

Thus, in addition to the kinase nucleotide sequences disclosed hereinand fragments and variants thereof, the isolated nucleic acid moleculesof the invention also encompass homologous DNA sequences identified andisolated from other cells and/or organisms by hybridization with entireor partial sequences obtained from the kinase nucleotide sequencesdisclosed herein or variants and fragments thereof.

The present invention also encompasses antisense nucleic-acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire kinase codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding a kinase protein. The noncoding regionsare the 5′ and 3′ sequences that flank the coding region and are nottranslated into amino acids.

Given the coding-strand sequences encoding a kinase protein disclosedherein (e.g., SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13), antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of kinase mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of kinase mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of kinase mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation proceduresknown in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example, phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or

genomic DNA encoding a kinase protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation. Anexample of a route of administration of antisense nucleic acid moleculesof the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,antisense molecules can be linked to peptides or antibodies to form acomplex that specifically binds to receptors or antigens expressed on aselected cell surface. The antisense nucleic acid molecules can also bedelivered to cells using the vectors described herein. To achievesufficient intracellular concentrations of the antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave kinase mRNA transcripts to thereby inhibittranslation of kinase mRNA. A ribozyme having specificity for akinase-encoding nucleic acid can be designed based upon the nucleotidesequence of a kinase cDNA disclosed herein (e.g., SEQ ID NO:1,3, 5, 7,9, 11, or 13). See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cechet al., U.S. Pat. No. 5,116,742. Alternatively, kinase mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, kinase gene expression can be inhibitedby targeting nucleotide sequences complementary to the regulatory regionof the kinase protein (e.g., the kinase promoter and/or enhancers) toform triple helical structures that prevent transcription of the kinasegene in target cells. See generally Helene (1991) Anticancer Drug Des.6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992)Bioassays 14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of a kinase molecule can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of the invention can also be used, e.g., in the analysis of singlebase pair mutations in a gene by, e.g., PNA-directed PCR clamping, asartificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (Hyrup (1996), supra, or as probes orprimers for DNA sequence and hybridization (Hyrup (1996), supra;Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of a kinase molecule can be modified, e.g.,to enhance their stability, specificity, or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup (1996), supra; Finn et al. (1996)Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res.17:5973; and Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

II. Isolated Kinase Proteins and Anti-kinase Antibodies

Kinase proteins are also encompassed within the present invention. By“kinase protein” is intended proteins having the amino acid sequence setforth in SEQ ID NO:2, 10 4, 6, 8, 10, 12, or 14, as well as fragments,biologically active portions, and variants thereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-kinaseantibodies. Fragments include peptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequences of akinase protein of the invention and exhibiting at least one activity ofa kinase protein, but which include fewer amino acids than thefull-length kinase proteins disclosed herein. Typically, biologicallyactive portions comprise a domain or motif with at least one activity ofthe kinase protein. A biologically active portion of a kinase proteincan be a polypeptide which is, for example, 10, 25, 50, 100 or moreamino acids in length. Such biologically active portions can be preparedby recombinant techniques and evaluated for one or more of thefunctional activities of a native kinase protein.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 45%, 55%, 65%, preferably about 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2, 4,6, 8, 10, 12, or 14. Variants also include variants of polypeptidesencoded by a nucleic acid molecule that hybridizes to a nucleic acidmolecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, or a complement thereofunder stringent conditions. Such variants generally retain thefunctional activity of the kinase proteins of the invention. Variantsinclude polypeptides that differ in amino acid sequence due to naturalallelic variation or mutagenesis.

The invention also provides kinase chimeric or fusion proteins. As usedherein, a kinase “chimeric protein” or “fusion protein” comprises akinase polypeptide operably linked to a non-kinase polypeptide. A“kinase polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a kinase protein, whereas a “non-kinasepolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein that is not substantially identical to thekinase protein, e.g., a protein that is different from the kinaseprotein and which is derived from the same or a different organism.Within a kinase fusion protein, the kinase polypeptide can correspond toall or a portion of a kinase protein, preferably at least onebiologically active portion of a kinase protein. Within the fusionprotein, the term “operably linked” is intended to indicate that thekinase polypeptide and the non-kinase polypeptide are fused in-frame toeach other. The non-kinase polypeptide can be fused to the N-terminus orC-terminus of the kinase polypeptide.

One useful fusion protein is a GST-kinase fusion protein in which thekinase sequences are fused to the C-terminus of the GST sequences. Suchfusion proteins can facilitate the purification of recombinant kinaseproteins.

In yet another embodiment, the fusion protein is a kinase-immunoglobulinfusion protein in which all or part of a kinase protein is fused tosequences derived from a member of the immunoglobulin protein family.The kinase-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a kinase ligand and a kinaseprotein on the surface of a cell, thereby suppressing kinase-mediatedsignal transduction in vivo. The kinase-immunoglobulin fusion proteinscan be used to affect the bioavailability of a kinase cognate ligand.Inhibition of the kinase ligand/kinase interaction may be usefultherapeutically, both for treating proliferative and differentiativedisorders and for modulating (e.g., promoting or inhibiting) cellsurvival. Moreover, the kinase-immnunoglobulin fusion proteins of theinvention can be used as immunogens to produce anti-kinase antibodies ina subject, to purify kinase ligands, and in screening assays to identifymolecules that inhibit the interaction of a kinase protein with a kinaseligand.

Preferably, a kinase chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences may be ligatedtogether in-frame, or the fusion gene can be synthesized, such as withautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments, whichcan subsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY).Moreover, a kinase-encoding nucleic acid can be cloned into acommercially available expression vector such that it is linked in-frameto an existing fusion moiety. Variants of the kinase proteins canfunction as either kinase agonists (mimetics) or as kinase antagonists.Variants of the kinase protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the kinase protein. An agonistof the kinase protein can retain substantially the same or a subset ofthe biological activities of the naturally-occurring form of the kinaseprotein. An antagonist of the kinase protein can inhibit one or more ofthe activities of the naturally-occurring form of the kinase protein by,for example, competitively binding to a downstream or upstream member ofa cellular signaling cascade that includes the kinase protein. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the kinase proteins.

Variants of the kinase protein that function as either kinase agonistsor as kinase antagonists can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the kinase proteinfor kinase protein agonist or antagonist activity. In one embodiment, avariegated library of kinase variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of kinase variants can be producedby, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential kinase sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of kinase sequences therein. There are avariety of methods that can be used to produce libraries of potentialkinase variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential kinase sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

In addition, libraries of fragments of the kinase protein codingsequence can be used to generate a variegated population of kinasefragments for screening and subsequent selection of variants of a kinaseprotein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double-stranded PCR fragment of a kinasecoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double-stranded DNA,renaturing the DNA to form double-stranded DNA which can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, one can derive an expression library thatencodes N-terminal and internal fragments of various sizes of the kinaseprotein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of kinase proteins. The mostwidely used techniques, which are amenable to high through-put analysisfor screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquethat enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify kinasevariants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated kinase polypeptide of the invention can be used as animmunogen to generate antibodies that bind kinase proteins usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length kinase protein can be used or, alternatively, theinvention provides antigenic peptide fragments of kinase proteins foruse as immunogens. The antigenic peptide of a kinase protein comprisesat least 8, preferably 10, 15, 20, or 30 amino acid residues of theamino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12, or 14 andencompasses an epitope of a kinase protein such that an antibody raisedagainst the peptide forms a specific immune complex with the kinaseprotein. Preferred epitopes encompassed by the antigenic peptide areregions of a kinase protein that are located on the surface of theprotein, e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-kinasepolyclonal and monoclonal antibodies that bind a kinase protein.Polyclonal anti-kinase antibodies can be prepared by immunizing asuitable subject (e.g., rabbit, goat, mouse, or other mammal) with akinase immunogen. The anti-kinase antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilized kinaseprotein. At an appropriate time after immunization, e.g., when theanti-kinase antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497, the humanB-cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72),the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodiesand Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-kinase antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a kinase protein to thereby isolateimmunoglobulin library members that bind the kinase protein. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant anti-kinase antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and nonhumanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNos. WO 86101533 and WO 87/02671; European Patent Application Nos.184,187, 171,496, 125,023, and 173,494; U.S. Pat. Nos. 4,816,567 and5,225,539; European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-kinase antibody (e.g., monoclonal antibody) can be used toisolate kinase proteins by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-kinase antibody canfacilitate the purification of natural kinase protein from cells and ofrecombinantly produced kinase protein expressed in host cells. Moreover,an anti-kinase antibody can be used to detect kinase protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the kinase protein. Anti-kinase antibodiescan be used diagnostically to monitor protein levels in tissue as partof a clinical testing procedure to, for example, determine the efficacyof a given treatment regimen. Detection can be facilitated by couplingthe antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs or

homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,

streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, .alpha.-interferon,.beta.-interferon,

nerve growth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.

475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a kinase protein(or a portion thereof). “Vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked, such as a “plasmid”, a circular double-stranded DNA loop intowhich additional DNA segments can be ligated, or a viral vector, whereadditional DNA segments can be ligated into the viral genome. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of genes to which they are operably linked. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors). However, the invention isintended to include such other forms of expression vectors, such asviral vectors (e.g., replication-defective retroviruses, adenoviruses,and adeno-associated viruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., kinase proteins, mutant formsof kinase proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of kinase protein in prokaryotic or eukaryotic host cells.Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or nonfusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRITS (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible nonfusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.), pp. 60-89). Strategies to maximize recombinant proteinexpression in E. coli can be found in Gottesman (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, CA),pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118.Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kujan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), particular promoters of T-cell receptors (Winotoand Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Baneiji etal. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Patent Publication No.264,166). Developmentally-regulated promoters are also encompassed, forexample the murine hox promoters (Kessel and Gruss (1990) Science249:374-379), the α-fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to kinase mRNA. Regulatory sequences operably linkedto a nucleic acid cloned in the antisense orientation can be chosen todirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen to direct constitutive,tissue-specific, or cell-type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid, or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986)Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboraty Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a kinase protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) kinase protein.Accordingly, the invention further provides methods for producing kinaseprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of the invention, into which arecombinant expression vector encoding a kinase protein has beenintroduced, in a suitable medium such that kinase protein is produced.In another embodiment, the method further comprises isolating kinaseprotein from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichkinase-coding sequences have been introduced. Such host cells can thenbe used to create nonhuman transgenic animals in which exogenous kinasesequences have been introduced into their genome or homologousrecombinant animals in which endogenous kinase sequences have beenaltered. Such animals are useful for studying the function and/oractivity of kinase genes and proteins and for identifying and/orevaluating modulators of kinase activity. As used herein, a “transgenicanimal” is a nonhuman animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA that is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous kinase gene has been alteredby homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingkinase-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The kinasecDNA sequence can be introduced as a transgene into the genome of anonhuman animal. Alternatively, a homologue of the mouse kinase gene canbe isolated based on hybridization and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thekinase transgene to direct expression of kinase protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan (1986)Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the kinase transgene in its genome and/orexpression of kinase mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding kinase gene can further be bred to other transgenicanimals carrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of a kinase gene or a homologue of thegene into which a deletion, addition, or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the kinasegene. In a preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous kinase gene is functionallydisrupted (i.e., no longer encodes a functional protein; such vectorsare also referred to as “knock out” vectors). Alternatively, the vectorcan be designed such that, upon homologous recombination, the endogenouskinase gene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous kinase protein). In thehomologous recombination vector, the altered portion of the kinase geneis flanked at its 5′ and 3′ ends by additional nucleic acid of thekinase gene to allow for homologous recombination to occur between theexogenous kinase gene carried by the vector and an endogenous kinasegene in an embryonic stem cell. The additional flanking kinase nucleicacid is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation), and cells in which the introducedkinase gene has homologously recombined with the endogenous kinase geneare selected (see, e.g., Li et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, ed.Robertson (IRL, Oxford), pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Current Opinion in Bio/Technology 2:823-829and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The kinase nucleic acid molecules, kinase proteins, and anti-kinaseantibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other

diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic or

inorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic or

inorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the ken of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small

molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a kinase protein or anti-kinase antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to

be achieved, and the limitations inherent in the art of compounding suchan active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express kinase protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect kinasemRNA (e.g., in a biological sample) or a genetic lesion in a kinasegene, and to modulate kinase activity. In addition, the kinase proteinscan be used to screen drugs or compounds that modulate cellular growthand/or metabolism as well as to treat disorders characterized byinsufficient or excessive production of kinase protein or production ofkinase protein forms that have decreased or aberrant activity comparedto kinase wild type protein. In addition, the anti-kinase antibodies ofthe invention can be used to detect and isolate kinase proteins andmodulate kinase activity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to kinase proteins or have a stimulatory or inhibitoryeffect on, for example, kinase expression or kinase activity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially-addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the kinaseprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the kinase protein or biologically active portion thereofcan be determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

In a similar manner, one may determine the ability of the kinase proteinto bind to or interact with a kinase target molecule. By “targetmolecule” is intended a molecule with which a kinase protein binds orinteracts in nature. In a preferred embodiment, the ability of thekinase protein to bind to or interact with a kinase target molecule canbe determined by monitoring the activity of the target molecule. Forexample, the activity of the target molecule can be monitored bydetecting induction of a cellular second messenger of the target (e.g.,intracellular Ca2+, diacylglycerol, IP3, etc.), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., a kinase-responsiveregulatory element operably linked to a nucleic acid encoding adetectable marker, e.g., luciferase), or detecting a cellular response,for example, cellular differentiation or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a kinase protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the kinase protein or biologicallyactive portion thereof. Binding of the test compound to the kinaseprotein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting thekinase protein or biologically active portion thereof with a knowncompound that binds kinase protein to form an assay mixture, contactingthe assay mixture with a test compound, and determining the ability ofthe test compound to preferentially bind to kinase protein orbiologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting kinase protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the kinase proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of a kinase protein can beaccomplished, for example, by determining the ability of the kinaseprotein to bind to a kinase target molecule as described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of a kinaseprotein can be accomplished by determining the ability of the kinaseprotein to further modulate a kinase target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting thekinase protein or biologically active portion thereof with a knowncompound that binds a kinase protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to or modulate theactivity of a kinase target molecule.

In the above-mentioned assays, it may be desirable to immobilize eithera kinase protein or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. In one embodiment, a fusionprotein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example,glutathione-S-transferase/kinase fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the nonadsorbed targetprotein or kinase protein, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtitre plate wellsare washed to remove any unbound components and complex formation ismeasured either directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of kinase binding or activity determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either kinaseprotein or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated kinase molecules or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96-well plates (Pierce Chemicals). Alternatively,antibodies reactive with a kinase protein or target molecules but whichdo not interfere with binding of the kinase protein to its targetmolecule can be derivatized to the wells of the plate, and unboundtarget or kinase protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the kinase protein or targetmolecule, as well as enzyme-linked assays that rely on detecting anenzymatic activity associated with the kinase protein or targetmolecule.

In another embodiment, modulators of kinase expression are identified ina method in which a cell is contacted with a candidate compound and theexpression of kinase mRNA or protein in the cell is determined relativeto expression of kinase mRNA or protein in a cell in the absence of thecandidate compound. When expression is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofkinase mRNA or protein expression. Alternatively, when expression isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of kinase mRNA or protein expression. The level of kinase mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting kinase mRNA or protein.

In yet another aspect of the invention, the kinase proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with kinase protein (“kinase-bindingproteins” or “kinase-bp”) and modulate kinase activity. Suchkinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins as, for example, upstreamor downstream elements of a signaling pathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial kinase gene sequences of the inventioncan be used to map their respective kinase genes on a chromosome,thereby facilitating the location of gene regions associated withgenetic disease. Computer analysis of kinase sequences can be used torapidly select PCR primers (preferably 15-25 bp in length) that do notspan more than one exon in the genomic DNA, thereby simplifying theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the kinasesequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map a kinasesequence to its chromosome include in situ hybridization (described inFan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries. Furthermore, fluorescence in situhybridization (FISH) of a DNA sequence to a metaphase chromosomal spreadcan be used to provide a precise chromosomal location in one step. For areview of this technique, see Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques (Pergamon Press, NY). The FISH technique canbe used with a DNA sequence as short as 500 or 600 bases. However,clones larger than 1,000 bases have a higher likelihood of binding to aunique chromosomal location with sufficient signal intensity for simpledetection.

Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results in a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the kinase gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The kinase sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes and probed on a Southern blot to yield unique bandsfor identification. The sequences of the present invention are useful asadditional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, thekinase sequences of the invention can be used to prepare two PCR primersfrom the 5□ and 3□ ends of the sequences. These primers can then be usedto amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The kinase sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. The noncoding sequences of SEQ ID NO:1, 3,5, 7, 9, 11, or 13 can comfortably provide positive individualidentification with a panel of perhaps 10 to 1,000 primers that eachyield a noncoding amplified sequence of 100 bases. If predicted codingsequences, such as those in SEQ ID NO:1, 3 5, 7, 9, 11, or 13 are used,a more appropriate number of primers for positive individualidentification would be 500 to 2,000.

3. Use of Partial kinase Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair, skin, or body fluids, e.g., blood, saliva, or semen found ata crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:1, 3, 5, 7, 9, 11, or 13 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include thekinase sequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:1, 3, 5, 7, 9, 11, or 13 having a lengthof at least 20 or 30 bases.

The kinase sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes that can beused in, for example, an in situ hybridization technique, to identify aspecific tissue. This can be very useful in cases where a forensicpathologist is presented with a tissue of unknown origin. Panels of suchkinase probes, can be used to identify tissue by species and/or by organtype.

In a similar fashion, these reagents, e.g., kinase primers or probes canbe used to screen tissue culture for contamination (i.e., screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting kinase protein and/or nucleic acid expression as well askinase activity, in the context of a biological sample. An exemplarymethod for detecting the presence or absence of kinase proteins in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting kinase protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes kinase protein such that the presence of kinaseprotein is detected in the biological sample. Results obtained with abiological sample from the test subject may be compared to resultsobtained with a biological sample from a control subject.

A preferred agent for detecting kinase mRNA or genomic DNA is a labelednucleic acid probe capable of hybridizing to kinase mRNA or genomic DNA.The nucleic acid probe can be, for example, a full-length or partialkinase nucleic acid, such as the nucleic acid of SEQ ID NO:1, 3, 5, 7,9, 11, or 13, or a portion thereof, such as a nucleic acid molecule ofat least 15, 30, 50, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions tokinase mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays of the invention are described herein.

A preferred agent for detecting kinase protein is an antibody capable ofbinding to kinase protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect kinase mRNA, protein, or genomic DNAin a biological sample in vitro as well as in vivo. For example, invitro techniques for detection of kinase mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetection of kinase protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations, and immunofluorescence.In vitro techniques for detection of kinase genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of kinaseprotein include introducing into a subject a labeled anti-kinaseantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. Biological samples may be obtained from blood, serum, cells, ortissue of a subject.

The invention also encompasses kits for detecting the presence of kinaseproteins in a biological sample (a test sample). Such kits can be usedto determine if a subject is suffering from or is at increased risk ofdeveloping a disorder associated with aberrant expression of kinaseprotein. For example, the kit can comprise a labeled compound or agentcapable of detecting kinase protein or mRNA in a biological sample andmeans for determining the amount of a kinase protein in the sample(e.g., an anti-kinase antibody or an oligonucleotide probe that binds toDNA encoding a kinase protein, e.g., SEQ ID NO:1, 3, 5, 7, 9, 11, or13). Kits can also include instructions for observing that the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of kinase sequences if the amount ofkinase protein or mRNA is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to kinaseprotein; and, optionally, (2) a second, different antibody that binds tokinase protein or the first antibody and is conjugated to a detectableagent. For oligonucleotide-based kits, the kit can comprise, forexample: (1) an oligonucleotide, e.g., a detectably labeledoligonucleotide, that hybridizes to a kinase nucleic acid sequence or(2) a pair of primers useful for amplifying a kinase nucleic acidmolecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of kinase proteins.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with kinase protein, kinasenucleic acid expression, or kinase activity. Prognostic assays can beused for prognostic or predictive purposes to thereby prophylacticallytreat an individual prior to the onset of a disorder characterized by orassociated with kinase protein, kinase nucleic acid expression, orkinase activity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and kinase protein or nucleic acid (e.g., mRNA,genomic DNA) is detected, wherein the presence of kinase protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant kinase expression oractivity. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid cell sample, or tissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreasekinase activity) to effectively treat a disease or disorder associatedwith aberrant kinase expression or activity. In this manner, a testsample is obtained and kinase protein or nucleic acid is detected. Thepresence of kinase protein or nucleic acid is diagnostic for a subjectthat can be administered the agent to treat a disorder associated withaberrant kinase expression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in a kinase gene, thereby determining if a subject with thelesioned gene is at risk for a disorder characterized by aberrant cellproliferation and/or differentiation. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion or mutation characterized by atleast one of an alteration affecting the integrity of a gene encoding akinase-protein, or the misexpression of the kinase gene. For example,such genetic lesions or mutations can be detected by ascertaining theexistence of at least one of: (1) a deletion of one or more nucleotidesfrom a kinase gene; (2) an addition of one or more nucleotides to akinase gene; (3) a substitution of one or more nucleotides of a kinasegene; (4) a chromosomal rearrangement of a kinase gene; (5) analteration in the level of a messenger RNA transcript of a kinase gene;(6) an aberrant modification of a kinase gene, such as of themethylation pattern of the genomic DNA; (7) the presence of anon-wild-type splicing pattern of a messenger RNA transcript of a kinasegene; (8) a non-wild-type level of a kinase-protein; (9) an allelic lossof a kinase gene; and (10) an inappropriate post-translationalmodification of a kinase-protein. As described herein, there are a largenumber of assay techniques known in the art that can be used fordetecting lesions in a kinase gene. Any cell type or tissue in whichkinase proteins are expressed may be utilized in the prognostic assaysdescribed herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the kinase-gene(see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). It isanticipated that PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a kinase gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns of isolated test sample and control DNA digested with one ormore restriction endonucleases. Moreover, the use of sequence specificribozymes (see, e.g., U.S. Pat. No. 5,498,531) can be used to score forthe presence of specific mutations by development or loss of a ribozymecleavage site.

In other embodiments, genetic mutations in a kinase molecule can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255;Kozal et al. (1996) Nature Medicine 2:753-759). In yet anotherembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence the kinase gene and detect mutations bycomparing the sequence of the sample kinase gene with the correspondingwild-type (control) sequence. Examples of sequencing reactions includethose based on techniques developed by Maxim and Gilbert ((1977) Proc.Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci.USA 74:5463). It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the diagnosticassays ((1995) Bio/Techniques 19:448), including sequencing by massspectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al.(1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl.Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the kinase gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). See, also Cotton et al. (1988) Proc. Natl. Acad. Sci. USA85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In apreferred embodiment, the control DNA or RNA can be labeled fordetection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in kinase cDNAs obtained from samples of cells.See, e.g., Hsu et al. (1994) Carcinogenesis 15:1657-1662. According toan exemplary embodiment, a probe based on a kinase sequence, e.g., awild-type kinase sequence, is hybridized to a cDNA or other DNA productfrom a test cell(s). The duplex is treated with a DNA mismatch repairenzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, e.g., U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in kinase genes. For example, single-strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild-type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double-stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a kinase gene.

3. Pharmacogenomics

Agents or modulators that have a stimulatory or inhibitory effect onkinase activity (e.g., kinase gene expression) as identified by ascreening assay described herein, can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant kinase activity as well as to modulate the cellular growth,differentiation and/or metabolism. In conjunction with such treatment,the pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of kinase protein, expression of kinase nucleic acid, ormutation content of kinase genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of kinase protein, expression of kinase nucleic acid,or mutation content of kinase genes in an individual can be determinedto thereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a kinase modulator, such as a modulator identified by one of theexemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of kinase genes (e.g., the ability to modulateaberrant cell proliferation and/or differentiation) can be applied notonly in basic drug screening but also in clinical trials. For example,the effectiveness of an agent, as determined by a screening assay asdescribed herein, to increase or decrease kinase gene expression,protein levels, or protein activity, can be monitored in clinical trialsof subjects exhibiting decreased or increased kinase gene expression,protein levels, or protein activity. In such clinical trials, kinaseexpression or activity and preferably that of other genes that have beenimplicated in for example, a cellular proliferation disorder, can beused as a marker of cellular growth and differentiation.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates kinase activity (e.g., as identified in ascreening assay described herein) can be identified. Thus, to study theeffect of agents on cellular proliferation disorders, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of kinase genes and other genes implicated inthe disorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of kinase genes or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent; (2) detecting the level of expression of a kinase protein, mRNA,or genomic DNA in the preadministration sample; (3) obtaining one ormore postadministration samples from the subject; (4) detecting thelevel of expression or activity of the kinase protein, mRNA, or genomicDNA in the postadministration samples; (5) comparing the level ofexpression or activity of the kinase protein, mRNA, or genomic DNA inthe preadministration sample with the kinase protein, mRNA, or genomicDNA in the postadministration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly to bring aboutthe desired effect, i.e., for example, an increase or a decrease in theexpression or activity of a kinase protein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant kinase expression oractivity. Additionally, the compositions of the invention find use inthe treatment of disorders described herein.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant kinaseexpression or activity by administering to the subject an agent thatmodulates kinase expression or at least one kinase gene activity.Subjects at risk for a disease that is caused, or contributed to, byaberrant kinase expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the kinase aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of kinase aberrancy, forexample, a kinase agonist or kinase antagonist agent can be used fortreating the subject. The appropriate agent can be determined based onscreening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating kinaseexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of kinase protein activity associated withthe cell. An agent that modulates kinase protein activity can be anagent as described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a kinase protein, a peptide, akinase peptidomimetic, or other small molecule. In one embodiment, theagent stimulates one or more of the biological activities of kinaseprotein. Examples of such stimulatory agents include active kinaseprotein and a nucleic acid molecule encoding a kinase protein that hasbeen introduced into the cell. In another embodiment, the agent inhibitsone or more of the biological activities of kinase protein. Examples ofsuch inhibitory agents include antisense kinase nucleic acid moleculesand anti-kinase antibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a kinaseprotein or nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or a combination of agents, that modulates (e.g.,upregulates or downregulates) kinase expression or activity. In anotherembodiment, the method involves administering a kinase protein ornucleic acid molecule as therapy to compensate for reduced or aberrantkinase expression or activity.

Stimulation of kinase activity is desirable in situations in which akinase protein is abnormally downregulated and/or in which increasedkinase activity is likely to have a beneficial effect. Conversely,inhibition of kinase activity is desirable in situations in which kinaseactivity is abnormally upregulated and/or in which decreased kinaseactivity is likely to have a beneficial effect.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXAMPLES

Gene Expression Analysis

Total RNA was prepared from various human tissues by a single stepextraction method using RNA STAT-60 according to the manufacturer'sinstructions (TelTest, Inc). Each RNA preparation was treated with DNaseI (Ambion) at 37° C. for 1 hour. DNAse I treatment was determined to becomplete if the sample required at least 38 PCR amplification cycles toreach a threshold level of flourescence using β-2 microglobulin as aninternal amplicon reference. The integrity of the RNA samples followingDNase I treatment was confirmed by agarose gel electrophoresis andethidium bromide staining. After phenol extraction cDNA was preparedfrom the sample using the SUPERSCRIPT™ Choice System following themanufacturer's instructions (GibcoBRL). A negative control of RNAwithout reverse transcriptase was mock reverse transcribed for each RNAsample.

Novel kinase expression was measured by TaqMan® quantitative PCR (PerkinElmer Applied Biosystems) in cDNA prepared from the following normalhuman tissues: lymph node, spleen, thymus, brain, lung, skeletal muscle,fetal liver, tonsil, colon, heart, and liver from one or two adultdonors; fibrotic liver samples prepared from two to seven differentdonors; resting and phytohemaglutinin activated peripheral bloodmononuclear cells (PBMC); CD3⁺, CD4⁺, and CD8⁺T cells; Th1 and Th2 cellsstimulated for six or 48 hours with anti-CD3 antibody; resting andlipopolysaccharide activated CD19⁺ B cells; CD34⁺ cells from mobilizedperipheral blood (mPB CD34⁺), adult resting bone marrow (ABM CD34⁺),G-CSF mobilized bone marrow (mBM CD34⁺), and neonatal umbilical cordblood (CB CD34⁺); G-CSF mobilized peripheral blood leukocytes (mPBleukocytes) and CD34⁻ cells purified from mPB leukocytes (mPB CD34⁻);CD14⁺ cells; and granulocytes. Transformed human cell lines includedK526, an erythroleukemia; HL60, an acute promyelocytic leukemia; Jurkat,a T cell leukemia; HEK 293, epithelial cells from embryonic kidneytransformed with adenovirus 5 DNA; and Hep3B hepatocellular livercarcinoma cells cultured in normal (HepB normoxia) or reduced oxygentension (Hep3B hypoxia).

Probes were designed by PrimerExpress software (PE Biosystems) based onthe sequence of each kinase gene. The primers and probes for expressionanalysis of h12832, h14138, h14833, h15990, h16341, and h2252,respectively, and for β-2 microglobulin were as follows: h12832 ForwardTTTTCACCTCCGACCTTTCCT Primer: h12832 Reverse ATCCCTTCCATTGTGAAAGCCPrimer: h12832 TaqMan CCAGGCGGTGAGACTCTGGACTGAG Probe: h14138 ForwardCACGAGGCTAGACTAAAAGGAAAATT Primer: h14138 Reverse TGAAGCCAGGAATACTGCTCAGPrimer: h14138 TaqMan TTGTGCTACAGACTAAATCCAGATACGGTCAG- Probe: GT h14833Forward CCTGCCTCCCACTCATCG Primer: h14833 ReverseCAGCTGGTTCTGTAGAGGACGAA Primer: h14833 TaqManATGCTCTGACTGCTCACTGCCTGGATC Probe: h15990 Forward GGCAAAGGCGGGTTCGPrimer: h15990 Reverse TTGACCGCCACATCGTAGC Primer: h15990 TaqManCCGGGCGCAACATAGGAAGTGG Probe: h16341 Forward CAGTTGCTAGACAGTAACCTGCATTTPrimer: h16341 Reverse TGAGGCCCACTGCTATGTCA Primer: h16341 TaqManCCTTGGACTGTGAGGGTAAAACTGGCC Probe: h2252 Forward TCTGATTCCGAGGGCTCTGAPrimer: h2252 Reverse ACGGTGGTAAAGTCTCATTCA Primer: h2252 TaqManCGGAATCTACCAGCAGGGAAAACAATACTCA Probe: T-C β-2 microglobulinCACCCCCACTGAAAAAGATGA Forward Primer β-2 microglobulinCTTAACTATCTTGGGCTGTGACAAAG Reverse Primer β-2 microglobulinTATGCCTGCCGTGTGAACCACGTG TaqMan Prober

Each kinase gene probe was labeled using FAM (6-carboxyfluorescein), andthe β2-microglobulin reference probe was labeled with a differentfluorescent dye, VIC. The differential labeling of the target gene andinternal reference gene thus enabled easurement in same well. Forwardand reverse primers and the probes for both β2-microglobulin and targetgene were added to the TaqMan® Universal PCR Master Mix (PE AppliedBiosystems). Although the final concentration of primer and probe couldvary, each was internally consistent within a given experiment. Atypical experiment contained 200 nM of forward and reverse primers plus100 nM probe for β-2 microglobulin and 600 nM forward and reverseprimers plus 200 nM probe for the target gene. TaqMan matrix experimentswere carried out on an ABI PRISM 7700 Sequence Detection System (PEApplied Biosystems). The thermal cycler conditions were as follows: holdfor 2 min at 50° C. and 10 min at 95° C., followed by two-step PCR for40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.

The following method was used to quantitatively calculate kinase geneexpression in the various tissues relative to β-2 microglobulinexpression in the same tissue. The threshold cycle (Ct) value is definedas the cycle at which a statistically significant increase inflourescence is detected. A lower Ct value is indicative of a highermRNA concentration. The Ct value of the kinase gene is normalized bysubtracting the Ct value of the β-2 microglobulin gene to obtain a_(Δ)Ct value using the following formula: _(Δ)Ct=Ct_(kinase)−Ct_(β-2)microglobulin. Expression is then calibrated against a cDNA sampleshowing a comparatively low level of expression of the kinase gene. The_(Δ)Ct value for the calibrator sample is then subtracted from _(Δ)Ctfor each tissue sample according to the following formula:_(ΔΔ)Ct=_(Δ)Ct-_(sample)−_(Δ)Ct-calibrator. Relative expression is thencalculated using the arithmetic formula given by 2^(−ΔΔCt). Expressionof the target kinase gene in each of the tissues tested is thengraphically represented as discussed in more detail below.

FIG. 22 shows expression of h12832 in various tissues and cell lines asdescribed above, relative to expression in CD14⁺ cells. The resultsindicate significant expression in thymus, fetal liver, B cells, Th1 andTh2 samples, and the K562, HL60, HEK 293, and Jurkat cell lines.

FIG. 23 shows expression of h14138 in various tissues and cell lines asdescribed above, relative to expression in mPB leukocytes. The resultsindicate broad tissue expression and significant expression in Th1 andTh2 cells, and the K562, HL60, HEK 293, and Jurkat cell lines.

FIG. 24 shows expression of h14833 in various tissues and cell lines asdescribed above, relative to expression in CD14⁺ cells. The results showbroad tissue expression with high levels in skeletal muscle, fetalliver, and tonsil. Significantly high expression is seen in the CD34⁺cells from mobilized peripheral blood and mobilized bone marrow, as wellas in colon, and the Jurkat and HEK 293 cell lines.

FIG. 25 shows expression of h15990 in various tissues and cell lines asdescribed above, relative to expression in HEK 293 cells. The resultsshow that expression of h15990 is broadly distributed among the tissuesexamined with a significantly high level of expression in colon.

FIG. 26 shows expression of h16341 in various tissues and cell lines asdescribed above, relative to expression in fibrotic liver cells (sampleNDR 194). The results indicate expression at low or barely detectablelevels in the tissues examined.

FIG. 27 shows expression of h2252 in various tissues and cell lines asdescribed above, relative to brain tissue. The results indicate thath2252 is expressed lymphocytic cells, particularly in the T and Blymphocyte subpopulations, as well as in the HEK 293 and Jurkat celllines.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:2; b) apolypeptide comprising a fragment of the amino acid sequence of SEQ IDNO:2, wherein the fragment has a kinase protein activity and comprisesat least 75 contiguous amino acids of SEQ ID NO:2; c) a polypeptidehaving a kinase protein activity, wherein said polypeptide is encoded bya nucleic acid molecule comprising a nucleotide sequence that is atleast 85% identical to SEQ ID NO:1, nucleotides 191-1156 of SEQ ID NO:1,or a complement thereof; and d) a polypeptide having a kinase proteinactivity, wherein the polypeptide is encoded by a nucleic acid moleculethat hybridizes to a nucleic acid molecule comprising SEQ ID NO:1,nucleotides 191-1156 of SEQ ID NO:1, or a complement thereof understringent conditions, said stringent conditions comprising hybridizationin 6×SSC at 42° C., followed by washing with 1×SSC at 55° C.
 2. Theisolated polypeptide of claim 1 comprising the amino acid sequence ofSEQ ID NO:2.
 3. The polypeptide of claim 1 further comprisingheterologous amino acid sequences.
 4. An antibody that selectively bindsto a polypeptide of claim
 1. 5. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 6. The polypeptide of claim 5further comprising heterologous amino acid sequences.
 7. An antibodythat selectively binds to the polypeptide of claim
 5. 8. A polypeptidecomprising a fragment of the amino acid sequence of SEQ ID NO:2, whereinthe fragment has a kinase protein activity and comprises at least 75contiguous amino acids of SEQ ID NO:2.
 9. The polypeptide of claim 8further comprising heterologous amino acid sequences.
 10. An antibodythat selectively binds to the polypeptide of claim
 8. 11. A polypeptidehaving a kinase protein activity, wherein said polypeptide is encoded bya nucleic acid molecule comprising a nucleotide sequence that is atleast 85% identical to SEQ ID NO:1, nucleotides 191-1156 of SEQ ID NO:1,or a complement thereof.
 12. The polypeptide of claim 11 furthercomprising heterologous amino acid sequences.
 13. An antibody thatselectively binds to the polypeptide of claim
 11. 14. A polypeptidehaving a kinase protein activity, wherein the polypeptide is encoded bya nucleic acid molecule that hybridizes to a nucleic acid moleculecomprising SEQ ID NO:1, nucleotides 191-1156 of SEQ ID NO:1, or acomplement thereof under stringent conditions, said stringent conditionscomprising hybridization in 6×SSC at 42° C., followed by washing with1×SSC at 55° C.
 15. The polypeptide of claim 14 further comprisingheterologous amino acid sequences.
 16. An antibody that selectivelybinds to the polypeptide of claim 14.